Mitosis is the process by which a replicated set of chromosomes is equally distributed between two daughter cells. Because cells require a complete genetic blueprint to function properly, it is essential for mitosis to occur without error mis-segregation of even a single chromosome can be the cause of genetic disease, cancer, and death. Chromosomes are segregated by a microtubule-based cellular machine termed the spindle. A key feature of the spindle is its bipolar geometry, an organization that naturally allow chromosomes to be segregated in two directions. Other organizational states, such as monopolarity or multipolarity, are largely incompatible with life and cause cell death via apoptosis. This renewal application is focused on how spindle bipolarity is both established and maintained. Spindle bipolarity in most eukaryotic cells is established by Eg5, a kinesin-5 motor. However, we discovered in the previous grant cycle that bipolarity in human cancer cells can be established through a novel Eg5-indpendent mechanism. This finding has important clinical implications, as kinesin-5 inhibitors (K5Is) have not performed well as anti-cancer agents in early stage clinical trials. Our work suggests that the inefficacy of K5Is may stem from alternative, compensatory spindle assembly pathways. Once the spindle has formed, it must remain bipolar despite the presence of forces that act in opposition to Eg5. The source, magnitude and temporal fluctuations of Eg5-opposing/assisting forces are not well-characterized, but our data implicate the involvement of kinetochore-attached microtubules (K- MTs). In this grant, we will: 1) Determine when and how K-MTs contribute to bipolarity maintenance; 2) Study features of a second kinesin (Kif15) key for Eg5-independent spindle assembly; and 3) Further characterize non-canonical spindle assembly mechanisms and their physiological shortcomings. This work will advance our understanding of spindle mechanics and have immediate relevance to the development of anti-mitotic chemotherapeutic strategies.